Gene Cry7ba1 Encoding and Insecticidal Crystal Protein of Bacillus Thuringiensis

The present invention discloses the isolation, cloning and use of insecticidal crystal proteins from Bacillus thuringiensis. The present invention isolates a novel insecticidal crystal protein gene cry7Bal from B. thuringiensis subsp. huazhongensis YBT-978 strain, and said gene encodes a novel insecticidal crystal protein Cry7Bal, which shows insecticidal activity against Lepidopteran insects. The present invention also discloses the gene sequence of the novel insecticidal crystal protein, uses suitable expression vectors to transform micro-organisms so as to express the product Cry7Bal encoded by the gene and make it exert the insecticidal activity of the protein against Lepidopteran pests.

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Description
FIELD OF THE INVENTION

The present invention relates to micro-organism genetic engineering. In particular, the present invention relates to the separation and cloning of insecticidal crystal proteins from Bacillus thuringiensis. The present invention relates to the genetic engineering of biological pesticides.

BACKGROUND OF THE INVENTION

The Bacillus thuringiensis is a rod-shaped, Gram-positive, endospore-forming bacterium which is widely existing in various ecological environments. During the spore-forming phase, B, thuringiensis forms parasporal crystals consisting of Insecticidal Crystal Proteins (ICPs) which have specific toxicity against insects and specific biological activities to more than 500 species of insects in 10 orders belonging to the class Insecta, including Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, etc, as well as to some harmful varieties in Protozoa, Nematomorpha, Platyhelminthes (Schnepf, H E., Crickmore, N., Rie, J. V., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D. R. & Dean, D. H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62, 775-806).

Wildtype strains of B. thuringiensis typically have genes encoding insecticidal crystal proteins and each strain usually has multiple copies of said genes. Schnepf et al. cloned the first gene encoding insecticidal crystal protein in 1981 from B. thuringiensis subsp. kurstah Strain HD1 and deduced the amino acid sequence of the first B. thuringiensis insecticidal crystal protein based on the DNA base sequence (Schnepf H E, Wong H C, Whiteley H R. The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA base sequence. J Biol. Chem. 1985 May 25; 260(10):6264-6272.). Afterwards, new genes encoding insecticidal crystal proteins have been actively pursued in connection with studies of B. thuringiensis. Many new genes encoding different insecticidal crystal proteins have been identified, cloned and sequenced. Therefore, in 1995, the B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee was founded by scholars including Crickmore in the Annual Meeting of the Society for Invertebrate Pathology. In 1996, a new classification system for the B. thuringiensis insecticidal crystal proteins based on the homology of amino acid sequences was formally proposed, and nomenclature rules and the principle for classification were set, wherein it stipulates that: cry gene is an insecticidal gene from B. thuringiensis encoding parasporal crystal protein, or any gene that has obvious sequence similarity to a known cry gene; cyt gene is a gene encoding a parasporal crystal protein from B. thuringiensis that exhibits hemolytic activity, or any gene encoding a protein that has obvious sequence similarity to a known Cyt protein. They are classified in 4 ranks based on the homology of the amino acid sequence deduced from the full length gene. The boundaries between each rank represent approximately 95%, 78% and 45% sequence identity. The genes of the insecticidal crystal protein are classified into 4 ranks. By August, 2005, the number of genes of Bacillus thuringiensis insecticidal crystal proteins has reached 319, representing varieties from 48 types (Crickmore, N., D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean. 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:807-813; see for example the internet site at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/index.html on the world wide web).

In the beginning, pesticides based on B. thuringiensis are produced with screened wildtype strains. With the advancement of molecular biology, people have gradually altered the wildtype strains by genetic engineering means. At the same time, people have been keeping on transforming genes of B. thuringiensis insecticidal crystal proteins into plants and have been producing transgenic plants that are resistant to agricultural pests.

However, with the development of B.t. pesticides and the increasing usage of these pesticides, resistance in targeted pests has been continuously discovered by the scientists. The scholars have extensively studied the resistance against B.t pesticides in targeted pests. Bacillus thuringiensis insecticidal crystal proteins have to go through the following process in order to give the insecticidal effects: the solubilization of the crystals and activation of the protoxin crystals, binding of the toxin fragments to receptors on the epithelial linings in the midgut, and the insertion into the membrane to create pores, wherein the activity spectrum and toxicity mainly depend on the recognition and interaction of the toxin fragments with the specific receptors on the epithelial linings in the midgut of the insects. Further, the development of resistance against B.t. pesticides in insects is closely related to the recognition and binding to the pesticide receptor. Therefore, cloning and application of new, especially novel genes of insecticidal crystal proteins have become the key to prevent and control the resistance against B.t pesticides in targeted pests and the core issue in the various insect control strategies. In recent years, searching and cloning novel genes of insecticidal crystal proteins have been the most active area in the study for B. thuringiensis. The significance of the present invention lies in this. In China, a new subspecies, YBT-978 strain, which is a subspecies of B. thuringiensis has been separated and characterized in 1996, which belongs to subspecies huazhongensis, serotype H40 (for the source of the strain, please refer to Dai J et al. 1996. Bacillus thuringiensis subsp. huazhongensis, serotype H40, isolated from soils in the People's Republic of China. Letters in Applied Microbiology. 22(1): 42-45). It is found the parasporal crystal proteins has a highly efficient insecticidal activity to insects including Plutella xylostella through extracting said parasporal crystal proteins and subjecting to bioassays.

CONTENTS OF THE INVENTION

The object of the present invention is to isolate and clone the genes of insecticidal crystal proteins having high toxic potency from B. thuringiensis and to provide a use for the same.

The technical scheme of the present invention is as shown in FIG. 1.

In order to achieve the aforesaid object, the present invention isolates from Bacillus thuringiensis subsp. huazhongensis, serotype H40, strain YBT-978 (for the source of the strain, please refer to Dai J et al. 1996. Bacillus thuringiensis subsp. huazhongensis, serotype H40, isolated from soils in the People's Republic of China. Letters in Applied Microbiology. 22(1): 42-45) a novel insecticidal crystal protein gene, the coding region of which consists of 3465 bases and has a nucleotide sequence shown in SEQ ID NO: 1. The cry7Ba1 gene of the present invention encodes Cry7Ba1 protein, which consists of 1154 amino acid residues and has an amino acid sequence as shown in SEQ ID NO:2. The gene cry7Ba1 of the present invention is expressed in microorganisms as Cry7Ba1 protein, which possesses insecticidal activity against insects of the order Lepidoptera.

The highest homology between the Cry7Ba1 protein of the present invention and any one of the proteins which has been disclosed is 58.2%. Moreover, the highest homology between the sequence of the N-terminal half of the Cry7Ba1 protein of the present invention (amino acid 1 to 658 of the amino acid sequence shown in SEQ ID NO:2) and any one of the proteins that has been disclosed is 37.1%.

The present invention provides the aforesaid gene sequence, which is a novel insecticidal crystal protein gene having insecticidal activity. The said gene can be used in transforming microorganisms and/or plants to impart insecticidal activity against pests of the Lepidoptera, as well as to overcome and delay the resistance in pests against genetically engineered insecticides and transgenic plants.

The following descriptions and examples disclose the technical solution in more detail:

It should be noted that the standard procedures and the reagents used on DNA are according to “Guide for Molecular Cloning Experiments” (refer to J. Sambrook et al (2002). Molecular Cloning: A Laboratory Manuel 3rd Edition. Translated by Jin Dong-yan et al. Science Press. Beijing).

1. The Sequencing of the N-Terminal Amino Acids of Crystal Protein Cry7Ba1 in B. thuringiensis YBT-978

The present invention uses B. thuringiensis YBT-978 as source strain for insecticidal crystal protein Cry7Ba1. B. thuringiensis YBT-978 is a standard strain of B. thuringiensis serotype H40, the source of which was recited in Dai J et al. 1996. The strain can form the typical rhombus-shaped crystals, and the molecular weight of the main component of the crystal protein Cry7Ba1 is about 130 kDa. The specific steps for sequencing the N-terminal amino acid sequence of Cry7Ba1 are as follows:

(1) The Culture of Bacterial Strains and the Manipulation of Culture

B. thuringiensis YBT-978 is inoculated on 5 ml LB (1% NaCl 1% peptone, 0.5% yeast extraction, pH 7.0) using an inoculation loop under sterile conditions and is incubated overnight in 30° C. on a shaker at 200 rpm. Then, 350 μl of the culture is transferred under sterile conditions to 35 ml ICPM culture medium (0.6% peptone, 0.5% glucose, 0.1% CaCO3, 0.1% MgSO4.7H2O, 0.05% KH2PO4, pH 7.0), and is incubated in 30° C. on a shaker at 200 rpm until gemma and parasporal crystals completely fall off. The culture is collected and is centrifuged for 1 minute at 8000 rpm. Discard the supernatant. Use 0.5% NaCl solution and sterilized de-ionized water to rinse the precipitates for 3 times separately.

(2) The SDS-PAGE Electrophoresis of Crystal Proteins and the Transfer of PVDF Blotting Membrane

Resuspend the rinsed precipitates in de-ionized water at a ratio of precipitates:de-ionized water=1:5 v/v, and add in an equal volume of 2× loading buffer. Mix, and then incubate in a boiling bath for 3 to 5 minutes, centrifuge at 12000 rpm for 5 minutes, and perform SDS-PAGE electrophoresis on the supernatant. For the formulation of 2× loading buffer and the procedure for performing SDS-PAGE electrophoresis, please refer to the method recited by Laemmli (Laemmli, UK. 1970. Digestion of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685).

Then, the protein samples on the gel are transferred to PVDF membrane using Trans-Blot SD Semi-Dry Transfer Cell (BIO-RAD) using standard method (see Cook RG. 1994. Amino acid sequence analysis of blotted proteins, p. 207-220. In B. S. DunBar (ed.), Protein Blotting: A practical approach. Oxford University Press, New York).

(3) Determination of the N-Terminal Amino Acid Sequence of the Crystal Protein

Use a protein N-terminal amino acid sequencer (model number:ABI 491 Protein Sequence, ABI Procise) to sequence the N-terminal amino acid sequence. The sequencing result is N-terminal-MDIRNQNKYEVVYPA-C-terminal (SEQ ID NO: 3).

2. Acquisition of the sequence of 5′ end DNA fragment of the insecticidal crystal protein gene cry7Ba1

Perform amplification using adaptor-ligation PCR. The principles and steps of adaptor-ligation PCR was described in Slebert P. D. et al. (Slebert P D et al. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Research 23:1087-1088). Specific steps are as follows:

1) Extraction of Total DNA from B. Thuringiensis YBT-978

B. thuringiensis YBT-978 is inoculated on 5 ml LB culture medium using an inoculation loop under sterile conditions and is cultured overnight in 30° C. on a shaker at 200 rpm. Then, 50 μl of the culture is transferred under sterile conditions to 5 ml fresh LB culture medium, and incubate under same conditions till logarithmic growth phase. Collect the bacteria after centrifugation at 5000 rpm for 3 minutes. Rinse once with 1 ml STE. Add 100 μl solution I and 10 μl lysozyme (50 mg/ml) and allow for action for 30 minutes or above. Add 200 μl 2% SDS, and incubate with a water bath at 50-60° C. for 30 minutes. Add 200 μl 5 mol/L NaCl and mix. Add 500 μl phenol/chloroform/isopentyl alcohol (25:24:1 v/v) and centrifuge for 5 minutes at 12000 rpm. Collect the supernatant and repeat the extraction once or twice. Transfer the upper layer DNA solution to Eppendorf tubes and then add equal volumes of 95% ethanol. Set in room temperature for 5 minutes, and then centrifuge for 5 minutes at 12000 rpm. Use 200 μl 70% ethanol to rinse the precipitates once and freeze-dry the precipitates, then dissolve the freeze-dried precipitates in 50 μl TE solution.

2) Enzymatic Digestion of the Total DNA with Restriction Endonuclease and Ligation with Adaptor

(1) Allow the prepared total DNA of Bacillus thuringiensis to be enzymatically digested by HpaI enzyme at 37° C. overnight (the enzymatic digestion system is 50 μl comprising about 1 μg total DNA); (2) Add 50 μl enzymatic digestion product into 250 μl phenol:chloroform:isopentyl alcohol (25:24:1 v/v), centrifuge at 12000 rpm for 5 minutes, transfer the upper layer DNA solution to Eppendorf tubes, add equal volumes of 95% ethanol, set at room temperature for 5 minutes, then centrifuge at 12000 rpm for 5 minutes. The precipitate is rinsed with 200 μl 70% ethanol once and then freeze-dried and dissolved in 50 μl sterilized water; (3) Mix equal volume of long and short adaptors (50 μM) (the sequence of long adaptor is: 5 GCTCGAGTCGACCGTGGTACGCGTGTGTGAGCTCCCGGGATCCGGT-3′ (SEQ ID NO: 4); the sequence of short adaptor is 5 ACCGGATC-NH2-3′) to give a 4 μl solution, which is incubated in 70° C. for 10 minutes; (4) 4 μl adaptor solution (25 μM), 10 μl the solution of the total DNA enzymatic digestion product (contains about 0.5 μg DNA), 8U T4 ligase, 2 μl T4 ligase buffer; and add water to make a 20 μl system. Ligate at 16° C. for 12 hours; (5) After heating at 60° C. for 5 minutes, the ligation system used as a template for PCR amplification;

3) Nested PCR and Sequencing

Two rounds of PCR amplification are carried out with the aforesaid ligation product.

Based on the sequenced N-terminal amino acid sequence, synthetic gene-specific primers which are abbreviated as 130P1 and 130P2 are designed. Moreover, synthetic adaptor primers abbreviated as AP1 and AP2 are designed based on the long adaptor sequence. The sequences of the primers are as follows:

130P1: 5-ATGGATATHMGNAATCARAAYAARTAYG-3 (SEQ ID NO:5) 130P2: 5-AATAAATATGARGTWGTNTAYCCNGC-3 (SEQ ID NO:6) AP1: 5-GCTCGAGTCGACCGTGGTA-3 (SEQ ID NO:7) AP2: 5-GTACGCGTGTGTGAGCTCC-3 (SEQ ID NO:8) (H = A, C, or T; M-A or C; N = A, G, C, or T; R = A or G; Y = C or T; W = A or T)

(A) First round PCR. 25 μl reaction system contains: 2.5 μl 10×PCR reaction buffer, 1 μl dNTP (2.5 mM each), 0.5 μl adaptor primer API (20 mM), 0.5 μl specific primer 130P1 (20 mM), 1 μl template (the above ligation product), 0.5 μl ExTaq enzyme. Add sterilized de-ionized water to make it to 25 μl. The PCR conditions are: 94° C., 1 minute for 1 cycle; 94° C., 1 minute, 52° C., 1 minute, 72° C., 1.5 minute for 25 cycles; 72° C., 5 minutes for 1 cycle. The first round PCR product is diluted 50-fold to give the amplification template for the second round of PCR.

(B) Second round PCR. 50 μl reaction system contains: 5 μl 10×PCR reaction buffer, 2 μl dNTP (2.5 mM each), 1 μl adaptor primer AP2 (20 mM), 1 μl specific primer 130P2 (20 mM), 1 μl template (the product of the first round of PCR), 1 μl ExTaq enzyme. Add sterilized de-ionized water to make it to 50 μl. The PCR reaction conditions are the same as aforesaid, except that the annealing temperature is 56° C.

(C) The products of the second round of PCR are recovered by PCR product recovering kit (purchased from Veta-Gene Company) and are then ligated to T-vector. The ligation product is used to transform E. coli E.DH5α. Perform PCR amplification to screen the transformants by using AP2 and 130P2 as primers and using single colony bacteria as the template. And sequence the exogenous fragments in positive transformant T-vector. The sequencing result is the sequence between position 19 to 491 of the nucleotide in SEQ ID NO:1.

Specific primers abbreviated as 130S1 and 130A1-2 are designed based on the above sequencing result. They are used to screen BAC genomic library containing the gene for crystal protein and various positive transformants, and size of the amplification product is 434 bp. The sequence of the primers are:

130S1-2: TTCTAATACAACATCAAAGTATCCACTC (SEQ ID NO:9) 130A1-2: GGTATCGTTCCAATACTAATTCTAAAC (SEQ ID NO:10)

3. The Cloning of Insecticidal Crystal Protein Gene cry7Ba1 of B. thuringiensis YBT-978

1) Construction of Genomic BAC Library of B. thuringiensis YBT-978 Bacterial Strain

Use BAC library vector pBeloBAC 11 (as shown in FIG. 2) to construct the genomic BAC library of B. thuringiensis YBT-978 according to the method disclosed by Luo and Wing (2003) (refer to Luo and Wing. 2003. An improved method for plant BAC library construction, p. 3-19. In Grotewold, Erich, Plant functional genomic: methods and protocols. Scientific and medical publishers,/Humana Press, Totowa, USA). That is, B. thuringiensis YBT-978 is allowed to grow in LB culture medium till mid-logarithmic growth phase, and then the bacteria is collected by centrifugation at 10000 rpm for 1 minute in clean and sterilized 50 ml centrifugation tubes. Add 1 ml TE buffer (1 mM EDTA, 10 mM Tris base, pH 8.0) to the collected bacteria, which is then lightly dispersed on an vibrator. Add about 40 ml TE buffer, set for 5 minutes and collect the bacteria by centrifugation at 10000 rpm for 1 minute. Set a 50 ml centrifugation tube containing the bacteria in warm water at 50° C. and add 1.5 ml TE25S (0.3 M sucrose, 25 mM EDTA, 25 mM Tris Base, pH 8.0), then mix well and add 1.5 ml 2% warmed gel (which is a low melting point gel and specialized for making embedding block, 0.1 g of such gel is dissolved in 5 ml TE25S). Mix well using an autopipette and add the mixture into molds for making embedding blocks, which are then cooled in a refrigerator at 4° C. to set the embedding blocks. The remaining steps, which include the enzymatic digestion of YBT-978 in the embedding blocks by HindIII and the recovery of enzymatic digestion products, the preparation of BAC library vector pBeloBAC11, the ligation of the enzymatic digestion products to the vectors and the qualification of the transformed DH10B and BAC libraries, are identical to the methods reported by Luo and Wing (2003).

2) Screening of BAC Genomic Library and Cloning of the Insecticidal Crystal Protein Gene cry7Ba1.

(A) Screening of BAC Genomic Library

PCR amplification is carried out by using 130S1 and 130A1 as primers and using BAC library single colony bacteria as the template. The reaction system is 25 μl, the formulation of which is identical to aforesaid. The reaction conditions are the same with aforesaid, except that the annealing temperature is 55° C. Positive clone EMB0491 is selected. Extract recombinant plasmid pBMB0491 according to the method disclosed by J. Sambrook et al. (J. Sambrook et al (2002). Molecular Cloning: A Laboratory Manual. 3rd Edition. Translated by Jin Dong-yan et al. Science Press. Beijing). Enzymatic digestion result indicates that pBMB0491 contains an about 60 kb fragment of YBT-978 genome.

(B) Acquisition of Insecticidal Crystal Protein Gene cry7Ba1

Use HindIII to complete enzymatically digest the YBT-978 genomic fragment in the positive clones. The enzymatic digestion product is ligated with cloning vector pUC18 (see FIG. 3) which is also complete enzymatically digested by HindIII. And the ligation product is used to transform E. coli DH5α. Primers numbered as 130S1-2 and 130A1-2 are again used as primers and transformant single colony bacteria is used as template to perform PCR amplification to screen positive transformant EMB0493, Extract recombinant plasmid pBMB0492 from EMB0493. Enzymatic digestion result indicates that pBMB0492 contains an about 16 kb fragment of YBT-978 genome. Further analysis showed the insecticidal crystal protein gene cry7Ba1 locates in the 5 kb XhoI fragment.

(C) Sequence Analysis on Insecticidal Crystal Protein Gene cry7Ba1 and Insecticidal Crystal Protein Cry7Ba1

Analyze the nucleotide sequence of 5 kb XhoI fragment in which the insecticidal crystal protein gene cry7Ba1 locates and obtain a sequence comprising 5235 bp, wherein 3465 bp are coding region. The coding sequence is shown as SEQ ID NO:1.

The said coding sequence may encode a polypeptide consisting of 1154 amino acids, i.e. insecticidal crystal protein Cry7Ba1, the molecular weight of which is deduced to be 130558 Da.

Searching and comparing this polypeptide with amino acid sequences of other known Cry and Cyt proteins in the GenBank gene database on the web at ncbi.nlm.nih.gov, it is discovered that, among all proteins publicly disclosed, the one which is closest to insecticidal crystal protein Cry7Ba1 is insecticidal crystal protein Cry7Ab2 (GenBank registration number: U04368), Cry7Ab1 (GenBank registration number: U04367) and CryAa1 (GenBank registration number: M64478), with the homology is 58.2%, 57.9% and 57.1% respectively. The homology of the sequence at the N-terminal half (position 1 to 658 amino acid in SEQ ID NO:2) is very low, which is the homology of 37.1%, 37.0% and 36.4% respectively. Said half is the part responsible for insecticidal activity and is essential and adequate for exerting insecticidal activity (Schnepf H E, Crickmore N, Rie J V, Lereclus D, Baum J, Feitelson J, Zeigler D R, Dean D H. 1998. B. thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev, 62:775-806). From this it can be seen the insecticidal gene is a novel insecticidal gene. Therefore, International B. thuringiensis Gene Nomenclature Committee named the said insecticidal crystal protein gene as cry7Ba1 which is determined to be a model gene of cry7B gene. Moreover, the sequence is highly similar to the known proteins at the C-terminal half (i.e. position 659 to 1154 amino acids of SEQ ED NO:2), with the homology reaching 87.9%, 86.9% and 87.9% respectively. The sequence of this part is the functional structural domain for forming the parasporal crystal, which is not relevant to insecticidal activity.

It can be seen from the above sequence analysis that, among all disclosed proteins, the homology of the one which is closest to the insecticidal crystal protein Cry7Ba1 of the present invention is 58.2%, which does not exceed 80%. When the homology of the amino acid sequence of a certain protein to the insecticidal crystal protein Cry7Ba1 exceeds 80%, it is deemed as falling within the scope of insecticidal crystal protein Cry7Ba1 of the present invention. Similarly, among all disclosed proteins, the homology of the one which is closest to the N-terminal half (position 1 to 658 amino acids of SEQ ID NO:2) of the insecticidal crystal protein Cry7Ba1 of the present invention is 37.1%, which does not exceed 50%. When the homology of amino acid sequence of a protein to the N-terminal half (position 1 to 658 amino acids of SEQ ID NO:2) of insecticidal crystal protein Cry7Ba1 exceeds 50%, it is deemed as falling within the scope of insecticidal crystal protein Cry7Ba1 of the present invention.

4. Insecticidal Activity of Insecticidal Crystal Protein Cry7Ba1 of B. Thuringiensis YBT-978

5 kb XhoI fragment in which cry7Ba1 locates is transferred to E. coli-B. thuringiensis shuttle vector pHT304 (see FIG. 4, for origin of the vector, please refer to Arantes O and Lereclus D. 1991. Construction of cloning vectors for B. thuringiensis. Gene 108:115-119) to give a recombinant plasmid pBMB0495 (see FIG. 5). Then, pBMB0495 is transformed to an acrystalliferous mutant of B. thuringiensis CryB strain (for the origin of the strain, please refer to Wu D, Federici BA. 1993. A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J. Bacteroil., 175:5276-5280) using well established electroporation method (Wu Lan, Sun Ming, Yu Ziniu. A New Resolution Vector with cry1Ac10 Gene Based on Bacillus thuringiensis Transposon Tn4430. Acta Mcrobiologica Sinica. 2000, 40:264-269) to give recombinant bacteria BMB-0502.

(1) Expression of Insecticidal Crystal Protein Cry7Ba1

Activate BMB0502 by incubating it in LB culture medium (erythromycin can be added at a final concentration of 20 μg/ml) overnight. Then, it is transferred to ICPM culture medium (erythromycin can be added at a final concentration of 20 μg/ml), incubate until gemma completely mature and fall off. Collect the bacteria and rinse them with 0.5% NaCl and sterilized de-ionised water separately for 3 times. Re-mix fee bacteria with water at the ratio bacteria:sterilized de-ionised water=1:5; then add an equal volume of 2× loading buffer and mix well. Incubate in a boiling water bath for 3 to 5 minutes, then centrifuge at 12000 rpm for 5 minutes. Perform SDS-PAGE electrophoresis with the supernatants. For the formulation of 2× loading buffer and the procedure for performing SDS-PAGE electrophoresis, please refer to the method recited by Laemmli (Laemmli, UK. 1970. Digestion of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685). As shown in FIG. 6, BMB0502 can form a 130 kDa crystal protein, the size of which is identical to YBT-978 of the starting strain, whilst negative control BMB0503 (which only contains the shuttle vector pHT304) does not form such a corresponding band.

(2) Crystal Morphology of Insecticidal Crystal Protein Cry7Ba1

Activate BMB0502 by incubating it in LB culture medium (erythromycin can be added at a final concentration of 20 μg/ml) overnight. Then, it is transferred to ICPM culture medium (erythromycin can be added at a final concentration of 20 μg/ml), incubate until gemma almost mature and fall off. Collect the bacteria and rinse them with sterilized de-ionised water once. Re-mix the bacteria with water at the ratio bacteria:sterilized de-ionised water=1:5. Prepare samples for transmission electron microscopy (TEM) and embed with Epon812. Make ultra-thin slices, perform transmission observation and photo taking at 80 kv. MBM0502 can form bi-pyramid shaped parasporal crystal which is shown in FIG. 7.

(3) Evaluation of Insecticidal Effect of Insecticidal Crystal Protein Cry7Ba1

Insecticidal effect of insecticidal crystal protein Cry7Ba1 is evaluated using third instar larvae of Plutella xylostella. Activate BMB0502 by incubating it in LB culture medium (erythromycin can be added at a final concentration of 20 μg/ml) overnight. Then, it is transferred to ICPM culture medium (erythromycin can be added at a final concentration of 20 μg/ml), incubate till gemma almost mature and fall off. Collect the bacteria and rinse them with sterilized de-ionised water once to give a parasporal crystal mixture. Perform bioassay using standard assay procedure (please refer to Shen Ju-qun, Wang Mian-ju, Yu Zi-niu (1990). Standard Procedure and Method for Bacillus thuringiensis formulations. Chinese Journal of Biological Control (Suppl.): 12-16). The bioassay is repeated once for each of the samples. Calculate LC50. The results showed the insecticidal crystal protein Cry7Ba1 exerted very high toxicity against Plutella xylostella (as shown in Table 1). This means the Cry7Ba1 protein cloned in the present invention shows very high insecticidal activity against insects of the Lepidoptera Order.

TABLE 1 Experimental results from bioassay of the Cry7Bal protein cloned in the present invention against Plutella xylostella LC50 value (95% Time Regression confidence limit) (Day) Material Regression equation coefficient (ng/ml) 3 BMB0502 y = 0.9928x + 0.9946 95.7 (93.9-97.5) 3.0334 5 BMB0502 y = 1.0554x + 0.9901 39.9 (38.2-41.6) 3.3103

DESCRIPTION OF THE DRAWINGS

FIG. 1: Technical flowchart of the present invention

FIG. 2: Construction chart of BAC vector pBleoBAC11 of the example of the present invention

FIG. 3: Construction chart of cloning vector pUC18 constructed in the present invention

FIG. 4: Construction chart of the shuttle vector constructed in the example of the present invention

FIG. 5: Recombinant plasmid pBMB0495 constructed in the example of the present invention

FIG. 6: SDS-PAGE electrophoresis analysis of Bacillus thuringiensis Cry7Ba1 parasporal crystal protein cloned in the present invention

    • M: molecular weight markers
    • 1: parasporal crystal protein of YBT-978 strain
    • 2: parasporal crystal protein of BMB0502 strain
    • 3: control BMB0503 strain

FIG. 7: Morphology of parasporal crystal produced by Bacillus thuringiensis YBT-978 and BMB0502 strains involved in the present invention (the line standard represents 1 μm)

    • A, B: parasporal crystal morphology of YBT-978 strain;
    • C, D: parasporal crystal morphology of BMB0502 strain.

 SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION

The followings are examples showing the present invention. It should be noted that the examples only demonstrate the present invention but by no means limit the present invention. The various experimental procedures involved are standard techniques in the field of the invention. For the contents that are not specifically explained, skilled persons in the art can refer to various reference books, scientific articles, relevant manuals and handbooks that are available prior to the application date of the present invention to carry out the said contents.

Example 1 The Cloning of Insecticidal Crystal Protein Gene cry7Ba1 of Bacillus thuringiensis YBT-978

According to the method disclosed by Luo and Wing (2003) (refer to Luo and Wing. 2003. An improved method for plant BAC library construction, p. 3-19. In Grotewold, Erich, Plant functional genomic: methods and protocols. Scientific and medical publishers,/Humana Press, Totowa, USA), BAC library vector pBeloBAC11 was used to construct genomic BAC library of Bacillus thuringiensis YBT-978 strain (for the source of the bacterial strain, please refer to Dai J et al. 1996. Bacillus thuringiensis subsp. huazhongensis, serotype H40, isolated from soils in the People's Republic of China, Letters in Applied Microbiology. 22(1): 42-45). Primers numbered as 130S1-2 and 130A1-2 were used as primers and BAC library single colony bacteria is used as the template to perform PCR amplification. Positive clone EMB0491 was selected. HindIII was used to complete enzymatically digest the YBT-978 genomic fragment in the positive clones. The enzymatic digestion product was ligated with cloning vector pUC18 which was also completely enzymatically digested by HindIII to transform E. coli DH5α. Primers numbered as 130S1-2 and 130A1-2 were again used as primers and transformant single colony bacteria is used as template to perform PCR amplification to screen positive transformant EMB0493. Recombinant plasmid pBMB0492 was extracted from EMB0493. Enzymatic digestion result revealed that pBMB0492 contains a fragment of about 16 kb of YBT-978 genome. Further analysis showed the insecticidal crystal protein gene cry7Ba1 locates in the 5 kb XhoI fragment.

Example 2 Sequence Analysis on Insecticidal Crystal Protein Gene cry7Ba1 of Bacillus thuringiensis YBT-978

The nucleotide sequence of the 5 kb XhoI fragment, in which the insecticidal crystal protein gene cry7Ba1 locates, was analyzed. The result was that it was a sequence comprising 5235 bp, wherein 3465 bp were the coding sequence. The coding sequence is shown as SEQ BD NO:1.

The said coding sequence might encode a polypeptide consisting of 1154 amino acids, the molecular weight of which was deduced to be 130558 Da.

Comparing this polypeptide with amino acid sequences of other known Cry and Cyt proteins, it was discovered that it was closest to Cry7Ab2, Cry7Ab1 and CryAa1, with the homology being 58.2%, 57.9% and 57.1% respectively. The homology of the sequence at the N-terminal half (position 1 to 658 amino acid in SEQ ID NO:1) was very low, with the homology being 37.1%, 37.0% and 36.4% respectively. As the polypeptide was similar to Cry7A insecticidal crystal protein, it was named by the International Bacillus thuringiensis Gene Nomenclature Committee as cry7Ba1.

Example 3 Expression of Insecticidal Crystal Protein Gene cry7Ba1

5 kb XhoI fragment, in which cry7Ba1 locates, was transferred to E. coli-B. thuringiensis shuttle vector pHT304 (for origin of the vector, please refer to Arantes O and Lereclus D. 1991. Construction of cloning vectors for Bacillus thuringiensis. Gene 108:115-119) to give recombinant plasmid pBMB0495. Then, pBMB0495 was transferred to an acrystalliferous mutant of Bacillus thuringiensis CryB strain (for the origin of the strain, please refer to Wu D, Federici BA. 1993, A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis. J. Bacteroil., 175:5276-5280) using well established electroporation method (Wu Lan, Sun Ming, Yu Ziniu. A New Resolution Vector with cry1Ac10 Gene Based on Bacillus thuringiensis Transposon Tn4430. Acta Microbiologica Sinica. 2000, 40:264-269) to give recombinant bacteria BMB-0502.

The above recombinant bacteria named as BMB0502 was activated by incubating it in LB culture medium (erythromycin could be added at a final concentration of 20 μg/ml) overnight. Then, it was transferred to ICPM culture medium (erythromycin could be added at a final concentration of 20 μg/ml), incubation was carried out until gemma completely matured and fell off. The bacteria was collected and rinsed with 0.5% NaCl and sterilized de-ionised water separately for 3 times. The bacteria was re-mixed with water at the ratio bacteria:sterilized de-ionised water=1:5, then an equal volume of 2× loading buffer was added and mixed well. The mixture was treated in a boiling water bath for 3 to 5 minutes, then centrifuged at 12000 rpm for 5 minutes. SDS-PAGE electrophoresis was performed for the supernatants. For the formulation of 2× loading buffer and the procedure for performing SDS-PAGE electrophoresis, please refer to the method recited by Laemmli (Laemmli, UK. 1970. Digestion of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685). As shown in FIG. 6, BMB0502 forms a 130 kDa crystal protein, the size of which was identical to YBT-978 of the starting strain, whilst the negative control BMB0503 (which only contained the shuttle vector pHT304) did not form such a corresponding band.

Example 4 Insecticidal Effect of Insecticidal Crystal Protein Cry7Ba1

Insecticidal effect of insecticidal crystal protein Cry7Ba1 was evaluated using third instar larvae of Plutella xylostella. BMB0502 was activated by incubating it in LB culture medium (erythromycin could be added at a final concentration of 20 μg/ml) overnight. Then, it was transferred to ICPM culture medium (erythromycin could be added at a final concentration of 20 μg/ml), incubation was carried out until gemma almost matured and fell off. The bacteria was collected and rinsed with sterilized de-ionised water once to give a parasporal crystal mixture. Bioassay was performed using standard assay procedure (please refer to Shen Ju-qun, Wang Mian-ju, Yu Zi-niu (1990). Standard Procedure and Method for Bacillus thuringiensis formulations. Chinese Journal of Biological Control (Suppl.): 12-16). The bioassay was repeated once for each of the samples. LC50 was calculated. The results showed the insecticidal crystal protein Cry7Ba1 exerted very high toxicity against Plutella xylostella. This means the Cry7Ba1 protein cloned in the present invention showed very high insecticidal activity against insects of the Lepidoptera, The effects achieved by carrying out the example was shown in Table 1.

Claims

1. An isolated insecticidal crystal protein gene from Bacillus thuringiensis, wherein said gene encodes a Cry7Ba1 protein having at least 80% homology to the amino acid sequence shown in SEQ ID NO:2.

2. The isolated insecticidal crystal protein gene from Bacillus thuringiensis of claim 1, wherein said gene comprises a nucleotide sequence shown in SEQ ID NO:1.

3. An isolated insecticidal crystal protein gene from Bacillus thuringiensis, wherein said gene encodes a Cry7Ba1 protein comprising an amino acid sequence having at least 50% homology to the partial sequence numbered 1 to 658 of SEQ ED NO:2.

4. A recombinant DNA construct comprising the insecticidal crystal protein gene of claim 1 or 3.

5. A micro-organism comprising the recombinant DNA construct of claim 4.

6. A Bacillus thuringiensis insecticidal crystal protein comprising an amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ED NO:2.

7. The Bacillus thuringiensis insecticidal crystal protein of claim 5, wherein said protein comprises an amino acid sequence shown in SEQ ID NO: 2.

8. A Bacillus thuringiensis insecticidal crystal protein comprising an amino acid sequence having at least 50% homology to the partial sequence numbered 1 to 658 of SEQ ID NO:2.

9. An insecticidal fragment of the amino acid sequence as shown in SEQ ID NO:2.

10. A plant cell transformed with a nucleotide sequence encoding the amino acid sequence of claim 9.

Patent History
Publication number: 20090130758
Type: Application
Filed: Oct 17, 2006
Publication Date: May 21, 2009
Patent Grant number: 7838293
Applicant: HUAZHONG AGRICULTURAL UNIVERSITY (Hubei)
Inventors: Ming Sun (Hubei), Ziniu Yu (Hubei), Shouwen Chen (Hubei), Zhenyu Zhang (Hubei), Lifang Ruan (Hubei), Suxia Guo (Hubei), Jingyuan Dai (Hubei), Lin Li (Hubei), Ziduo Liu (Hubei)
Application Number: 12/089,992